KR20040063290A - Preparation method of Anemarrhena asphodeloides Bunge extracts and pharmaceutical composition for diabetes therapeutics using thereof - Google Patents

Abstract

PURPOSE: Provided is an extract of Anemarrhena asphodeloides Bunge which involves the activity of lowering the blood sugar, and is well adapted for use in treating the diseases related to diabetes, and in making food additives. CONSTITUTION: A method of making the extract of Anemarrhena asphodeloides Bunge comprises the steps of: extracting Anemarrhena asphodeloides Bunge at 0-100 deg.C with a solvent selected from water, polar organic solvent or a mixture thereof; removing the solid component from the extracted solution through centrifuging or filtering; and removing the extracting solvent from the extracted solution, followed by purifying it. The polar organic solvent is short chain alcohol with C1-C4.

Description

Preparation method of diabetes extract and pharmaceutical composition for treating diabetes-related diseases containing the extract {Preparation method of Anemarrhena asphodeloides Bunge extracts and pharmaceutical composition for diabetes therapeutics using consider}

The present invention relates to a method for preparing Anemarrhena asphodeloides Bunge extract and a pharmaceutical composition for treating diabetes-related diseases containing the extract, wherein the step is extracted with water, a polar organic solvent or a mixed solvent thereof, from the extract By removing the solids and removing the extraction solvent from the extract to obtain the extract of the hair extract to obtain the extract having a hypoglycemic effect, containing it to prepare a composition for treating diabetes-related diabetes, having a good hypoglycemic effect An extract and a pharmaceutical composition for treating diabetes-related diseases containing the extract.

According to the evidence report: clinical guideline on the identification, evaluation, and treatment of overweight and obesity in adults (1999, NIH), the National Institutes of Health (NIH) About 15.7 million people have type 2 diabetes.

In addition, according to the World Health Organization, the number of diabetics is estimated to be about 300 million by 2025, and type 2 diabetic patients who have lost insulin-controlling blood glucose function now account for about 5.9% of the US population. (Current Pharmaceutical Design, 2001, 7: 417-450). Rapidly increasing diabetes is one of chronic diseases that causes enormous economic and social damage.

Diabetes is a disease associated with carbohydrate metabolism characterized by hyperglycemic symptoms in the blood and can be classified into type 1 diabetes (insulin dependent diabetes mellitus) and type 2 diabetes (insulin independent diabetes). Type 1 diabetics have little or no ability to secrete in vivo insulin. These patients develop extreme hyperglycemia and are prone to ketosis and ketoacidosis. Type 2 diabetics have insulin secretion but are either at low levels or unbalanced due to excessive secretion of adverse hormones such as glucagon. It is known to be caused by insulin resistance, which is caused by dysregulated insulin secretion of ground state and stimulation, increased endogenous glucose production in the liver, and decreased glucose availability in peripheral insulin sensitive tissues. Patients with type 2 diabetes also develop hyperglycemia, but are not as sensitive to ketoemia or ketoacidosis as patients with type 1 diabetes.

Insulin-independent patients, who make up the majority of diabetics, use oral hypoglycemic agents in combination with diet for the treatment of diseases. Drugs used as hypoglycemic agents can be divided into three groups according to the method of lowering blood glucose. The first group stimulates the beta cells of the pancreas to promote insulin secretion. The sulfonyl urea and meglitinide drugs are used. The second group is biguanide and thia There is an insulin sensitizer represented by a zolidinedion-based drug. The third group of drugs inhibits the breakdown of sugars and inhibits the absorption of intestinal sugars. There are drugs such as acarbose and aglytol. Preferred hypoglycemic agents should be able to express their action rapidly to prevent excessive blood sugar rise after meals, and then to lose their effects in a short time without causing hypoglycemia, and also to improve metabolic symptoms accompanying diabetes. In this regard, the drugs are completely undesirable, and in order to adequately treat diabetes at home and abroad, it is necessary to develop oral hypoglycemic agents with less side effects and safety.

In order to solve the above problems, the inventors of the present invention extract the hairs with water, a polar organic solvent or a mixed solvent thereof, remove the solids from the extracts, and remove the extract solvents from the extracts. By obtaining the glyme extract having a hypoglycemic effect through the obtaining step, by containing it to prepare a composition for treating diabetes-related, the present invention was completed.

Accordingly, an object of the present invention is to provide a method for preparing the extract of Jimi having an excellent hypoglycemic effect and a pharmaceutical composition for treating diabetes-related diseases containing the extract according to the method.

1 is a graph showing the blood glucose change rate measured after oral and intraperitoneal administration of the hair extract of Example 1 to Lepr ^db / Lepr ^db mice.

Figure 2 is a graph showing the blood glucose change rate measured after intraperitoneal administration of the hair extract extract prepared in Example 2 to Lepr ^db / Lepr ^db mice.

Figure 3 is a graph showing the blood glucose change rate measured after intraperitoneal administration of the MC and EA fraction of the hair extract extract prepared in Example 3 to Lepr ^db / Lepr ^db mice.

The present invention is 1) extracting the hair with an extraction solvent of water, polar organic solvent or a mixed solvent thereof, 2) removing the solids from the extract obtained in the extraction step, and 3) from the extract Characterized by a method for producing a hair extract comprising three steps of removing the extraction solvent to obtain a hair extract.

The present invention is another feature of the pharmaceutical composition for treating diabetes-related diseases, including the hair extract according to the above method and a pharmaceutically acceptable carrier.

In another aspect, the present invention is characterized by another food additive having a hypoglycemic effect, including the hair extract in the method.

Referring to the present invention in more detail as follows.

In the present invention, the hair to be extracted means the rhizome of the liliaceae plant hair. The medicinal herb is called dried rhizome, and the peeled dry rhizome is classified as hair growth but the hair that can be used in the present invention includes both of these. The present invention relates to a method for extracting such hair, and also relates to a pharmaceutical composition and a food additive containing a hair extract having a hypoglycemic effect.

The preparation method of the hair extract according to the present invention will be described in more detail as follows.

First, the first step is to extract the hair with an extraction solvent consisting of water, a polar organic solvent or a mixed solvent thereof. As the polar organic solvent used as the extraction solvent, a lower alcohol having 1 to 4 carbon atoms, more preferably methanol or ethanol, may be used. In addition, a polar organic solvent having an appropriate polarity may be used. Such polar organic solvents may include dichloromethane (also called methylene chloride), acetone, ethyl acetate, acetonitrile and the like. These extraction solvents may be used alone or in combination.

Extraction may be performed according to conventional methods. Extraction is usually carried out by putting the hair and the extraction solvent in a suitable container and stirring. Such extraction may be carried out at a temperature of 0 to 100 ° C., preferably 50 to 80 ° C., for a time such that the extract can be sufficiently extracted, typically for 1 hour to 1 month. At this time, if the extraction temperature is increased, the extraction time can be shortened, but if the temperature is too high, the active ingredient may be decomposed or changed by a chemical reaction, it is good to maintain a suitable temperature.

Next, it is two steps to remove solids from the extract obtained in the extraction step. The solids may be removed by centrifugation or filtration, and the extract, which is the filtrate from which the solids are removed, is used in the next step.

In addition, the extraction solvent is removed from the extraction solution in three steps. In this way, the extract of the present invention can be obtained by removing the extraction solvent from the extract from which the solid content by filtration has been removed. At this time, removal of the extraction solvent may also be performed by a conventional method. Extraction solvent removal method may include exposure to vacuum evaporation, freeze drying, dry nitrogen or dry air by atmospheric evaporation, vacuum centrifugal evaporator and the like. Heat may or may not be applied depending on conditions upon removal of the extraction solvent, but excessive heat is not preferred.

The present invention may further comprise the step of purifying the extract so that the blood glucose lowering effect of the hair extract obtained by the three steps is more excellent. Purifying the extract may include dissolving or suspending the extract in water, contacting the aqueous solution or aqueous suspension with a polar organic solvent or a non-polar organic solvent which is not mixed with water, and distributing the components of the extract, and the polarity. Recovering a solution of the organic solvent or the aqueous solution, and removing the polar organic solvent or water therefrom to obtain a partially purified hair extract. In this case, the polar organic solvent is dichloromethane, chloroform, 1-butanol, 1-pentanol, 1-hexanol, cyclohexanol, 1-dodecanol, diethyl ether, tetrahydrofuran, ethyl acetate, ethyl acetoacetate Ethyl benzoate or a mixed solvent thereof may be used, and as the nonpolar organic solvent, n-hexane, cyclohexane, n-pentane, n-octane, petroleum ether, toluene or a mixed solvent thereof may be used.

In more detail, the purification step uses a difference in partition coefficient according to polarity and solubility characteristics so that the active ingredient having a hypoglycemic effect is included in the hair extract at a higher rate. That is, the extract is active fractionated with a polar organic solvent or a nonpolar organic solvent. In more detail, in order to remove the inactive ingredients that are well soluble in the nonpolar solvent, the hair extract obtained by the extraction process is first completely dissolved or suspended in water. At this time, in order to completely dissolve the extract in water, a large amount of water is required or heated, but it is accompanied by inconvenience of handling, so the next step may be performed even if the extract is not completely dissolved. Next, the non-polar organic solvent which is not mixed with water is brought into contact with the extract aqueous solution or the aqueous suspension. This process can be carried out in a fractionator. Then, an inactive ingredient having nonpolar characteristics contained in the extract aqueous solution or the water suspension is dissolved in the nonpolar organic solvent. Thereafter, the solution of the non-polar organic solvent is removed and water is removed from the aqueous solution to obtain a partially purified hair extract of the present invention.

As the non-polar organic solvent that can be used in the present invention, it is generally preferable to use an organic solvent having a polarity of less than petroleum ether in the polarity table provided for the fraction and not mixed with water. Particularly preferred nonpolar solvent is hexane. In general, the polarity order of the organic solvents shown in the polarity table provided for fractionation is as follows.

Cyclohexane <n-pentane <Hexane <Isooctane <Petrolether <Xylol <Isopropyl ether <Toluene <Diethyl ether <Chloroform <Dichloromethane <Tetrahydrofuran <Acetone <Dioxane <Ethyl acetate < n-propanol <n-butanol <ethanol <methanol <water (listed in nonpolar to polar order).

In addition, in order to remove the inactive ingredients well soluble in water, a polar organic solvent which is not mixed with water is contacted with the aqueous solution or aqueous suspension of the extract of the extract obtained in the extraction process in the same manner as described above. Then, an inactive ingredient that is well soluble in water is more distributed in water, while an active ingredient that is well soluble in polar organic solvent is more distributed in polar organic solvent. Therefore, by removing the aqueous solution and removing the polar organic solvent from the solution of the polar organic solvent, a partially purified hair extract can be obtained. By this process, it is possible to remove the inactive ingredients well soluble in water from the hair extract obtained by the extraction process.

As the polar organic solvent that can be used in the present invention, it is preferable to use an organic solvent or a mixed solvent thereof, which generally has a polarity of at least diethyl ether and at most butanol in the polarity table provided for fractionation, and is not mixed with water. It is preferable to use. Particularly preferred polar organic solvents are dichloromethane, ethyl acetate and mixed solvents thereof.

The removal of the inactive ingredients well dissolved in the non-polar organic solvent described above and the removal of the inactive ingredients well dissolved in water are preferably carried out sequentially, and any one may be performed as necessary. In addition, in order to remove the ineffective ingredients that are well soluble in water, the hair extract may be dissolved in a polar organic solvent and then contacted with water.

Since the extract of the present invention obtained by the method as described above is excellent in hypoglycemic effect, it can be usefully used in pharmaceutical compositions and food additives for the treatment and prevention of diabetes-related diseases.

That is, the pharmaceutical composition for treating diabetes-related diseases of the present invention is an active ingredient having a hypoglycemic effect, and includes a hair extract extracted from hair. Such hair extracts are typically formulated in combination with a carrier. The pharmaceutical composition of the present invention may be used in the form of solids such as particles or tablets, and may also be used in liquid form.

In addition, in the preparation of a pharmaceutical composition containing a hair extract of the present invention, the composition prepared by mixing or diluting the hair extract with a carrier, an excipient and a diluent is encapsulated in a carrier in the form of a capsule or other container or the like. It is preferable. Such formulations may be, for example, in the form of tablets, pills, granules, powders, elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, sterile powders and the like. In particular, the form formulated as a preparation for injection or oral administration is preferred.

Examples of suitable carriers, excipients and diluents for use in the pharmaceutical compositions of the present invention include aluminum salts, phenoxyethylethanol, water, physiological saline, lactose, dextrose, sorbitol, mannitol, calcium silicate, cellulose, methyl cellulose, Microcrystalline cellulose, polyvinylpyrrolidone, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In addition, the formulation of the pharmaceutical composition of the present invention may further comprise a filler, anti-coagulant, lubricant, wetting agent, fragrance, emulsifier, preservative and the like.

The dosage of the pharmaceutical composition for treating diabetes-related diseases of the present invention may be appropriately selected according to the sex, age, health condition, type of diabetes-related disease to be treated, severity, complications, etc. The daily dose for) is 0.5 to 3 g, preferably 1 to 2 g.

On the other hand, the composition comprising the hair extract or partially purified hair extract of the present invention does not necessarily mean that the drug is approved, it can be understood as a concept that includes a conventional functional food or even health supplements.

Hereinafter, the present invention is specifically illustrated through specific examples (and experimental examples). However, the following examples are merely illustrative of specific embodiments of the present invention, and are not intended to limit the scope of the present invention.

Example 1 Extraction of Antihyperglycemic Active Material from Hair

To get an active extract with hypoglycemic effect from the hair, 3 kg was immersed twice in 20 L of 50% ethanol twice for 4 hours. It was run at 80 ° C. for effective extraction and stirred using a stirrer. After 4 hours the solids were allowed to settle and the cotton wool was filtered out of the funnel to remove the final float. The filtrate was evaporated with a rotary evaporator and the amount of extract obtained was measured and recovered with 500 mL of tertiary distilled water per 100 g of extract.

Example 2 Fraction of Antihyperglycemic Active Materials from Hair

The extract recovered with water as in Example 1 was mixed with the same amount of butanol (Butanol, Jin chemicals pharmaceutical Co. Ltd., Korea) and put into a separation funnel (separation funnel), and left to stand for 24 hours after stirring only the upper butanol layer Was separated. The solvent was re-distilled 2 to 3 times more, and the butanol fraction and the remaining water layer were concentrated using a distillator, and then the solvent was completely removed in a vacuum oven and dried.

Each fraction was powdered for animal experiments and stored at -20 ° C.

Example 3 Subdivision of Antihyperglycemic Active Materials from Hair

The extract recovered with water as in Example 1 was first mixed with the same amount of hexane (n-Hexane, Jin chemicals pharmaceutical Co. Ltd., Korea) and placed in a separation funnel, allowed to stand for 24 hours after stirring Only the upper hexane layer was separated. The mixture was re-divided two to three times with hexane, the hexane fractions were concentrated using a distillator, and then the solvent was completely removed in a vacuum oven and dried.

The remaining water layer was secondly mixed with the same amount of methylene chloride (methylene chloride, Jin chemicals pharmaceutical Co. Ltd., Korea, hereinafter MC) and placed in a separator, left for 24 hours after stirring to separate only the MC layer of the lower layer. The mixture was re-distilled 2-3 times with MC, the MC fraction was concentrated using a distillator, and then dried by completely removing the solvent in a vacuum oven.

The remaining water layer was thirdly mixed with the same amount of ethyl acetate (Ethyl acetate, Jin chemicals pharmaceutical Co. Ltd., Korea, hereinafter EA) and placed in a separator, and left for 24 hours after stirring to separate only the upper EA layer. The fractions were re-distilled 2-3 more times with EA, the EA fractions were concentrated using a distillation, and then dried by completely removing the solvent in a vacuum oven.

The remaining water layer was fourthly mixed with the same amount of butanol (butanol, Jin chemicals pharmaceutical Co. Ltd., Korea) and placed in a separator, and left for 24 hours after stirring to separate only the upper butanol layer. The solvent was re-divided 2-3 times with butanol, and the butanol fraction was concentrated using a distillator and then dried by completely removing the solvent in a vacuum oven.

Finally, the remaining water layer was concentrated using a distillation, and then dried by removing moisture in a vacuum oven.

Each fraction was powdered for animal experiments and stored at -20 ° C.

Experimental Example 1. Lepr ^db / Lepr ^db Postprandial Blood Glucose Lowering Effect in Mice

In order to measure the hypoglycemic activity of the extract prepared by the above example, a diabetic-induced mouse was selected as a test animal and the glycemic effect was tested.

[Experiment animal setting]

Lepr ^db / Lepr ^db mice, the world's most widely used obese type diabetic animals, were used to test anti-diabetic and obesity effects in vivo. Lepr ^db / Lepr ^db mice do not regulate food intake due to mutations in the leptin receptor gene, an appetite suppressant, resulting in excessive nutrient accumulation and metabolic abnormalities in the body, leading to obese diabetes. It is known to be suitable for in vivo activity assays for antidiabetic candidates because it shows very high hyperglycemia with obesity.

Measurement of Blood Glucose by Preparation and Administration of Samples

The hair extracts prepared in Examples 1 to 3 were diluted with distilled water or 1% dimethyl sulfoxide (DMSO) according to the respective concentrations, and then orally (PO) or intraperitoneally once a day in Lepr ^db / Lepr ^db mice. (IP) for 10-12 days. The number of animals in each group was assigned to 5 animals, and the breeding room temperature was 22 ± 2, 55 ± 5% humidity and 12 hours of light and dark. After administration of the sample, the weight of the mouse was measured, and the blood obtained by partially removing the tail was directly measured by a glucometer (Glucometer, Roche, Germany). The results are shown in Figs.

1 shows the blood glucose change rate measured after oral administration (P.O) and intraperitoneal administration (I.P) of the extract of Example 1, which was confirmed to be excellent in the hypoglycemic effect in both administration methods.

Figure 2 shows that the blood glucose change rate measured after intraperitoneal administration of the hair extract prepared in Example 2 showed a significant hypoglycemic effect, Figure 3 MC and the hair extract of the hair extract prepared in Example 3 The change in blood glucose measured after intraperitoneal administration of the EA fraction showed significant hypoglycemic effect on days 5 and 10.

Experimental Example 2. Toxicity Test of ICR Mice with Hair Extracts

In order to measure the toxicity of the extract prepared by the above example, 4-5 week old ICR mice fasted for 1 day were selected as the test animals.

The hair extract prepared according to the above example was diluted with distilled water or 1% DMSO (dimethyl sulfoxide) according to each administration concentration, and then intraperitoneally administered in four doses in a multiple of three. The number of animals in each group was assigned to 10 animals, and the LD ₅₀ value calculated based on this was 600 mg / kg for the EA layer of Example 3 and 500 mg / kg for the MC layer of Example 3. It became.

As described above, the hair extract according to the present invention exhibits hypoglycemic activity as a herbal extract, and thus can be usefully used as a therapeutic agent for diabetes and food additives.

Claims (11)

1) 1 step of extracting gimo with an extraction solvent consisting of water, a polar organic solvent or a mixed solvent thereof, 2) removing two solids from the extract obtained in the extraction step, and 3) 3 steps to remove the extract solvent from the extract solution to obtain a hair extract Method of producing a hair extract, characterized in that it comprises a. The method of claim 1, wherein the polar organic solvent is a lower alcohol having 1 to 4 carbon atoms. The method of claim 1, wherein the extraction of the first step is carried out at a temperature of 0 ~ 100 ℃. The method of claim 1, wherein the removal of solids in the two steps removes solids by centrifugation or filtration to obtain a filtrate. The method of claim 1, further comprising the step of purifying the hair extract obtained by the three steps. The method of claim 5, wherein the purification step of the extract Dissolving or suspending the extract in water, Dispensing the components of the extract by contacting the aqueous solution or aqueous suspension with a polar or non-polar organic solvent that is not mixed with water, and Recovering the solution or the aqueous solution of the polar organic solvent, and removing the polar organic solvent or water therefrom to obtain a partially purified hair extract Method of producing a hair extract, characterized in that it comprises a. The method of claim 6, wherein the polar organic solvent is dichloromethane, chloroform, 1-butanol, 1-pentanol, 1-hexanol, cyclohexanol, 1-dodecanol, diethyl ether, tetrahydrofuran, ethyl acetate, Method for producing a hair extract, characterized in that selected from ethyl acetoacetate, ethyl benzoate and a mixed solvent thereof. The method of claim 6, wherein the nonpolar organic solvent is selected from n-hexane, cyclohexane, n-pentane, n-octane, petroleum ether, toluene, and a mixed solvent thereof. A pharmaceutical composition for treating diabetes related diseases, comprising: a hair extract obtained from the method of any one of claims 1 to 8 and a pharmaceutically acceptable carrier. The pharmaceutical composition according to claim 9, wherein the pharmaceutical composition is formulated as a preparation for injection or oral administration. A food additive having a hypoglycemic effect, comprising a hair extract obtained by the method of any one of claims 1 to 8.